Project Summary/Abstract Processed meat is an established risk factor for colorectal cancer (CRC), but underlying mechanisms are still unknown and intake in the US remains high. Further, the burden of cancer and other adverse health outcomes attributable to high processed meat intake has been estimated to contribute to over 644,000 deaths in 2013 worldwide. Hypothesized key mechanisms involve additives in processed meats such as nitrate or nitrite, precursors to carcinogenic N-nitroso compounds (NOCs), which are alkylating agents and can induce G > A transitions. Due to the lack of a long-term biomarker for NOC exposure, previous studies relating dietary intake of nitrate or NOCs to CRC have been inconsistent. Despite the dearth of scientific evidence directly linking N- nitroso compounds (NOC) per se to human cancers, processed meat items labeled ?no nitrates/nitrites added? are increasingly being sold as a ?healthy? alternative to traditional processed meats leading many consumers to believe that it is perfectly ?safe? to consume ?healthy? processed meat items. However, there is evidence from a limited number of studies that higher processed meat intake may be associated with CRC driver genes with higher frequency of G>A transitions, indicating a mechanistic link with NOCs. Recent advances in next generation high output sequencing have facilitated the assessment ?mutational signatures? which are defined by the type of DNA damage resulting from certain DNA damaging agents, e.g., alkylating damage due to NOC exposure. We propose a very cost-effective study by using existing data from the Nurses' Health Study I (NHS) established in 1976 with 121,701 female nurses aged 30- 55 years at enrollment and the Health Professionals Follow-Up Study (HPFS) established in 1986 with 51,529 male health professionals aged 40-75 years. Self- administered questionnaires collected detailed data on lifestyle (updated every two years) and diet (updated every four years). Tumor tissues are also available in these cohorts. We will integrate repeated measures of diet and tumor tissue whole exome sequencing data (WES) to study underlying pathways. Aim 1a will assess whether cases with high processed meat intake have a higher frequency of G>A transitions in known and novel driver genes. Aim 1b will examine whether high processed meat consumers are more likely to exhibit a mutational signature indicative of alkylating DNA damage in their exome than those with low processed meat intake. Examining WES data in the context of our prospectively collected diet data provides an unprecedented opportunity to gain more insight into hypothesized or novel pathways and strengthen biologic plausibility. This knowledge is crucial to inform dietary guidelines for cancer prevention and guide food reformulation efforts. While avoiding processed meat is undoubtedly preferable, changing consumer choices is extremely time consuming. In the meantime, identifying interventions that can inhibit processed meat toxicity may be more effective to lessen the burden of cancers attributable to high processed meat consumption.